Novel Analogues of Antimicrobial and Anticancer Peptide Synthesized and Produced from Gaegurin 5

ABSTRACT

The present invention is related to antimicrobial and anticancer peptide engineering using Gaegurin 5 isolated from Korean frog ( Rana rugosa ), which have smaller structure compared with previously known Gaegurin peptide and shows potent antimicrobial and anticancer activity. Specifically, the antimicrobial and anticancer peptide of the present invention synthesized from the shortest length of Gaegurin 5, show potent antimicrobial activity against gram positive and negative strains, good safety with very low hemolytic activity and favorable advantages such as drug absorption and drug transportation due to its advantageous structural property, which can be useful as a potent antimicrobial or anticancer agent.

TECHNICAL FIELD

The present invention is related to analogues of antimicrobial andanticancer peptide synthesized and produced from Gaegurin 5

BACKGROUND ART

Antimicrobial peptide acting on cellular membrane has been found in mostof all the species in the world at now. Recently, the antimicrobialpeptide has been paid attention to researchers to overcome the problemsof conventional antibiotics, i.e., the increase of antibiotic resistantbacteria. Especially, after the first finding of bombinins from Bombinavariegate in 1969, the skin of anurans (frogs and toads) has proven tobe a rich source of antimicrobial peptides with a broad-spectrum ofantimicrobial activities. After the discovery of antimicrobial peptidefrom African toenail frog, i.e., magainins in 1987, the frog skinantimicrobial peptide has been increasingly focused as potentialtherapeutic agents.

Since antimicrobial peptides kill bacteria by acting on bacteria cellmembrane and destroying the membrane selectively, the mechanism ofantimicrobial peptide is quitely different from that of the existingantibiotics and is valuable as an alternative proposal for overcomingresistance problems. Furthermore, since the antimicrobial peptides havebroad spectrum of antimicrobial activities for gram-positive microbe,gram-negative microbe, fungus, virus and tumor cell, the naturalsubstance isolated from natural resource is expected to be a goodantibiotic showing no side effect. Additionally, since it showsamphipathic property, i.e., soluble in both of water and lipid, it isexpected to have great advantages in respect to drug absorption, drugtransportation etc. However, in spite of the favorable advantages ofantimicrobial peptides, there remain several problems such as structuralstability, bulky M. W. etc, in developing antimicrobial peptide as adrug, the big problems of antimicrobial antibiotics are stability andmolecular weight as follows: First, in aspect of stability, it is easilydecomposed since the antimicrobial peptides could not resist to lots ofprotein lyases existing in vivo. Those problems can be solved byintroducing unnatural derived amino acid such as D-amino acid,beta-amino acid, and modifying their chemical structures and so on.However, another problem, i.e., the bulky size of antimicrobial peptidehaving M. W. more than 3,000 still remains to solve the problems inrespect to drug absorption, drug transportation etc.

Anticancer agent can be classified into three categories, i.e.,bio-engineered drug such as anticancer drug using gene, enzyme, vaccineetc, synthesized drug and natural product-derived drug. However, thereremain several problems, for example, most of bio-engineered drugs havenot been developed in clinical anticancer agent; and manychemotherapeutic agents have diverse pharmacological mechanism inaccordance with the type of cancer (Gillman et al., The pharmacologicalBasis of therapeutics, Maxwell Macmillan., 18, p 1202, 1986) and toxicside effects (Chung et al., J. Wonkwang Medical ScL, 3, pp 13-34, 1987).Specifically, anticancer agent shows toxic effect not only on cancercell but also on normal cells and shows the resistance to anticanceragent caused by several factors, i.e., the mutation in process growth,proliferation and metastasis of cancer cells. Since most of anticanceragent have M. W. less than 1,000 Dalton, the administered anticanceragents were absorbed in cancer cell as well as normal tissue resultingin several damages of normal cell, especially, actively cell-dividingnormal cell, for example, dysfunction of bone marrow, gastrointestinaldisorder, alopecia etc. Due to its low M. W, it is easily excretedthrough urine therefore lots amount of agents are required to obtaindesirable medical effect.

Therefore, the present inventors have endeavored to overcome thepreviously reported problems of antibiotic peptide and anticancer agentand studied to find effective and novel peptide showing more potentefficacy till now. Finally, they have found that novel synthesizedanalogues based on Gaegurin 5, the smallest length among six kinds ofantimicrobial peptides previously designated as Gaegurin isolated fromKorean frog, i.e., Gaegurins 1 to 6, showed potent antimicrobial,anticancer and non-hemolytic activity.

DISCLOSURE OF INVENTION Technical Problem

The present invention provides novel analogues of antimicrobial andanticancer peptide synthesized and produced from Gaegurin 5 showingpotent antimicrobial and anticancer activity with broad spectrum andlittle hemolytic side effect, and the pharmaceutical compositioncomprising the same for the treatment and prevention of infectivediseases caused by microorganism and cancer diseases.

Technical Solution

Accordingly, it is an object of the present invention to provide novelanalogues of antimicrobial and anticancer peptide represented by Seq. I.D. 1 (F-L-G-W L F K W-A-K-K, hereinafter designated as “model 25”, Seq.I. D. 2 (F-L-K-W-L-F-K-W-A-K-K, hereinafter designated as “model 26”,Seq. I. D. 3 (F-L-G-W L F K W-A-W-K hereinafter designated as “model 27”and Seq. I. D. 4 (F-L-W-W-L-F-K-W-A-W-K, hereinafter designated as“model 28” synthesized and produced from Gaegurin 5.

The above-described peptide is synthesized by the method characterizedin substituting specific moiety of the peptide with tryptophan andlysine.

It is an object of the present invention to provide a pharmaceuticalcomposition comprising the above described antimicrobial and anticancerpeptides synthesized and produced from Gaegurin 5 as an effectiveingredient and pharmaceutically acceptable carrier or adjuvant for thetreatment and prevention of infective diseases caused by microorganismand cancer diseases.

It is an object of the present invention to provide a method of treatingor preventing infective diseases caused by microorganism and cancerdiseases of human and mammals comprising administering an effectiveamount of the above described antimicrobial and anticancer peptidesynthesized and produced from Gaegurin 5 with a pharmaceuticallyacceptable carrier thereof.

Additionally, the present invention also provide a use of thecomposition comprising the above described antimicrobial and anticancerpeptides synthesized and produced from Gaegurin 5, for the manufactureof a medicament for infective diseases caused by microorganism andcancer diseases in a mammal, together with a pharmaceutically acceptablecarrier thereof.

The term “infective diseases caused by microorganism” disclosed hereincomprises staphylococcus food poisoning, cellulitis, urinary tractinfection, meningitis, peritonitis, cystitis, lymphangitis, felon,tympanitis, respiratory disease, pneumonia, purulent inflammation,sepsis and the like, preferably, staphylococcus food poisoning,respiratory disease or pneumonia.

The term “cancer diseases” disclosed herein comprises cervical cancer,lung cancer, pancreas cancer, non-small cell lung cancer, liver cancer,colon carcinoma, cancer of a bone, skin cancer, cancer of the head andneck, cutaneous or intraocular melanoma, uterine carcinoma, ovariancancer, rectal cancer, stomach cancer, cancer of the anal region, breastcancer, oviduct cancer, endometrial carcinoma, vaginal cancer, vulvacancer, Hodgkin's disease, esophageal cancer, small intestine tumor,endocrine gland's cancer, thyroid cancer, parathyroid cancer, epinephroscancer, soft tissue sarcomas, urethrophyma, penis cancer, prostaticcarcinoma, bladder cancer, kidney and ureter cancer, preferably,cervical cancer, lung cancer, liver cancer, colon carcinoma, skincancer, stomach cancer, prostatic carcinoma or kidney cancer. Further,the composition of the present invention is useful in treatingmetastasis other than the above-described cancer diseases.

Hereinafter, the present invention is described in detail.

The inventive analogues of antimicrobial and anticancer peptidessynthesized and produced from Gaegurin 5 can be prepared in detail byfollowing procedures;

The antimicrobial and anticancer peptide of the present invention can besynthesized and prepared through modifying the structure of Gaegurin 5having the shortest residue (24 residues) selected from the six kinds ofpeptides (Gaegurin 1 to Gaegurin 6) isolated from Korean frog, i.e.,Rana rugosa.

Specifically, to synthesize optimized antimicrobial and anticancerpeptide analogues, the peptide analogue can be synthesized by solidphase synthetic method using Fmoc amino group protecting vessel,Gaegurins 4 and 5 as parent structures.

Preferably, the peptide analogue of the present invention can beprepared by the step consisting of: eliminating C-terminal residue ofGaegurin 4 side by side with screening its antibiotic activity; removingC-terminal 14 residues to the extent that their antibiotic activitiesreach to be disappeared in case that only 23 residues at N-terminalremains; substituting the residue positioned at 16 in inactive Gaegurin4 analogues with tryptophan residue to synthesize Gaegurin 4 analogues(D16W-GGN4N23) showing antibiotic activity, of which result confirmedthat the introduction of tryptophan thereto is a crucial requirement.

With the identical method with the above-described method for Gaegurin4, the antibiotic activity is screened with eliminating C-terminalresidue of Gaegurin 5 side by side, of which result confirmed thatminimal requirement for the activity is the analogue having 13 residues.As we described the above, since the introduction of tryptophan iscrucial in peptide engineering development, the tryptophan residue maybe introduced in inactive 11-residue fragment instead of active13-residue fragment of Gaegurin 5 to afford 11 kinds of tryptophansubstituted Gaegurin 5 analogues. At the result of identifying thebioactive activity of the analogues, tryptophan introduced novel twoanalogues, i.e., A4W-GG511 introduced at 4-position and V8W-GGN5N11introduced at 8-position showed similar activities to those of Gaegurin5. Furthermore, whether the introduction of only tryptophan into4-position and 8-position is specific or not, the introduction of theother amino acids, preferably, Leucine having hydrophobic residue,Lysine having hydrophilic and cationic ions, and Phenylalanine havingaromatic ring similar to tryptophan have been performed to synthesizenovel seven kinds of amino acid substitutes derived from Gaegurin 5.

To increase the potency of antimicrobial and anticancer activity ofA4W-GG511 and V8W-GGN5N11 analogues prepared by the above-describedmethod, novel modeling peptide analogues could be prepared by increasingthe number of tryptophan and substituting the residue positioned betweenhydrophilic end side and hydrophobic starting side with lysine.

Additionally, the antimicrobial and anticancer peptide analogues of thepresent invention could be prepared by methods including the abovedescribed synthetic method as well as genetic recombinant techniquemethod, for example, gene clone including DNA or RNA sequence coding forthe peptide which is suitable to express the above describedantimicrobial and anticancer peptide is prepared; and the gene clone istransformed into appropriate cells expressing peptide to obtain thepurposed antimicrobial and anticancer peptide of the present invention.

The present invention also provides antimicrobial and anticancer peptideanalogues prepared by the above described methods.

Additionally, the present invention provides a pharmaceuticalcomposition comprising the above-described antimicrobial and anticancerpeptide analogues synthesized and produced from Gaegurin 5 as aneffective ingredient and pharmaceutically acceptable carrier or adjuvantfor the treatment and prevention of infective diseases caused bymicroorganism and cancer diseases.

To investigate the most optimized antibiotic peptide analogues inrespect to its molecular weight and stability among the peptidesprepared by the above-described preparation method, the antimicrobial,anticancer and hemolytic activity of those analogues were examined andthe results indicated that following peptide analogues showed similarantimicrobial, anticancer and hemolytic activity to A4W-GG511 andV8W-GGN5N11 analogues synthesized and produced from Gaegurin 5:

model 25: F-L-G-W-L-F-K-W-A-K-K model 26: F-L-K-W-L-F-K-W-A-K-K model27: F-L-G-W-L-F-K-W-A-W-K model 28: F-L-W-W-L-F-K-W-A-W-K

In a while, following peptide analogue showed potent hemolytic activityin spite of its similar antimicrobial and anticancer activity toGaegurin 5:

W4, 8-GGN5N13: F-L-G-W-L-F-K-W-A-S-K-V-L

After monitoring various factors such as whether the antimicrobial andanticancer peptide analogues show structurally similarity to Gaegurin 5and favorable advantages in respect to antimicrobial, anticancer andhemolytic activity, it has been confirmed that minimal number of residueamong 23 residues in amino acid to satisfy purposed effects is 11 andthe analogues should show amphipathic property and the substitution atposition-4 and 8 shows the most highly increased activity. Additionally,the substitution of more than two tryptophan residues is required toobtain potent antimicrobial and anticancer activity however it causes torelatively increased hemolytic activity and further the substitution ofthe residues positioned at the amphipathic boundary surface of thepeptide with Lysine can be obtain increased activity.

Hereinafter, the following formulation methods and excipients are merelyexemplary and in no way limit the invention.

The composition according to the present invention can be provided as apharmaceutical composition containing pharmaceutically acceptablecarriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acaciarubber, alginate, gelatin, calcium phosphate, calcium silicate,cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineraloil. The formulations may additionally include fillers,anti-agglutinating agents, lubricating agents, wetting agents, flavoringagents, emulsifiers, preservatives and the like. The compositions of theinvention may be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after their administration to a patientby employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolvedin oils, propylene glycol or other solvents which are commonly used toproduce an injection. Suitable examples of the carriers includephysiological saline, polyethylene glycol, ethanol, vegetable oils,isopropyl myristate, etc., but are not limited to them. For topicaladministration, the compounds of the present invention can be formulatedin the form of ointments and creams.

Pharmaceutical formulations containing present composition may beprepared in any form, such as oral dosage form (powder, tablet, capsule,soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet,granule), or topical preparation (cream, ointment, lotion, gel, balm,patch, paste, spray solution, aerosol and the like), or injectablepreparation (solution, suspension, emulsion).

The composition of the present invention in pharmaceutical dosage formsmay be used in the form of their pharmaceutically acceptable salts, andalso may be used alone or in appropriate association, as well as incombination with other pharmaceutically active compounds.

The desirable dose of the inventive composition varies depending on thecondition and the weight of the subject, severity, drug form, route andperiod of administration, and may be chosen by those skilled in the art.However, in order to obtain desirable effects, it is generallyrecommended to administer at the amount ranging 0.01-1 Og/kg,preferably, 1 to 5 g/kg by weight/day of the inventive analogues of thepresent invention. The dose may be administered in single or dividedinto several times per day. In terms of composition, the compositionshould be present between 0.01 to 80% by weight, preferably 0.5 to 50%by weight based on the total weight of the composition.

The pharmaceutical composition of present invention can be administeredto a subject animal such as mammals (rat, mouse, domestic animals orhuman) via various routes. All modes of administration are contemplated,for example, administration can be made orally, rectally or byintravenous, intramuscular, subcutaneous, intracutaneous, intrathecal,epidural or intra-cerebroventricular injection.

The present invention is more specifically explained by the followingexamples. However, it should be understood that the present invention isnot limited to these examples in any manner.

ADVANTAGEOUS EFFECTS

The analogues of antimicrobial and anticancer peptide synthesized andproduced from Gaegurin 5 of the present invention show potentantimicrobial activity against gram positive and negative strains,potent anticancer activity against eight kinds of cancer cell lines,good safety with very low hemolytic activity and favorable advantagessuch as drug absorption and drug transportation due to theiradvantageous structural properties, i.e., the shortest structure amongpreviously known antimicrobial and anticancer peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings.

FIG. 1 shows the side view of the whole GGN5 structure in SDS micelles.

FIG. 2 shows the top view of the N-terminal part (G3-V13) in theamphipathic alpha-helix region in SDS micelles.

FIG. 3 depicts the helical wheel diagrams of model peptide 25.

FIG. 4 depicts the helical wheel diagrams of model peptide 26.

FIG. 5 depicts the helical wheel diagrams of model peptide 27.

FIG. 6 depicts the helical wheel diagrams of model peptide 28.

FIG. 7 presents the anticancer activity of model peptide 25 againstSK-MEL-2 of cancer cell lines.

FIG. 8 presents the anticancer activity of model peptide 25 against A549of cancer cell lines.

FIG. 9 presents the anticancer activity of model peptide 25 againstSK-OV-3 of cancer cell lines.

FIG. 10 presents the anticancer activity of model peptide 25 againstHCT116 of cancer cell lines.

FIG. 11 presents the anticancer activity of model peptide 25 againstMKN45 of cancer cell lines.

FIG. 12 presents the anticancer activity of model peptide 25 againstPC-3 of cancer cell lines.

FIG. 13 presents the anticancer activity of model peptide 25 againstA498 of cancer cell lines.

FIG. 14 presents the anticancer activity of model peptide 26 againstSK-MEL-2 of cancer cell lines.

FIG. 15 presents the anticancer activity of model peptide 26 againstA549 of cancer cell lines.

FIG. 16 presents the anticancer activity of model peptide 26 againstSK-OV-3 of cancer cell lines.

FIG. 17 presents the anticancer activity of model peptide 26 againstHCT116 of cancer cell lines.

FIG. 18 presents the anticancer activity of model peptide 26 againstMKN45 of cancer cell lines.

FIG. 19 presents the anticancer activity of model peptide 26 againstPC-3 of cancer cell lines.

FIG. 20 presents the anticancer activity of model peptide 26 againstA498 of cancer cell lines.

FIG. 21 presents the anticancer activity of model peptide 27 againstSK-MEL-2 of cancer cell lines.

FIG. 22 presents the anticancer activity of model peptide 27 againstA549 of cancer cell lines.

FIG. 23 presents the anticancer activity of model peptide 27 againstSK-OV-3 of cancer cell lines.

FIG. 24 presents the anticancer activity of model peptide 27 againstHCT116 of cancer cell lines.

FIG. 25 presents the anticancer activity of model peptide 27 againstMKN45 of cancer cell lines.

FIG. 26 presents the anticancer activity of model peptide 27 againstPC-3 of cancer cell lines.

FIG. 27 presents the anticancer activity of model peptide 27 againstA498 of cancer cell lines.

FIG. 28 presents the anticancer activity of model peptide 28 againstSK-MEL-2 of cancer cell lines.

FIG. 29 presents the anticancer activity of model peptide 28 againstA549 of cancer cell lines.

FIG. 30 presents the anticancer activity of model peptide 28 againstSK-OV-3 of cancer cell lines.

FIG. 31 presents the anticancer activity of model peptide 28 againstHCT116 of cancer cell lines.

FIG. 32 presents the anticancer activity of model peptide 28 againstMKN45 of cancer cell lines.

FIG. 33 presents the anticancer activity of model peptide 28 againstPC-3 of cancer cell lines.

FIG. 34 presents the anticancer activity of model peptide 28 againstA498 of cancer cell lines.

FIG. 35 represents the anticancer activity of A4W, V8W-GGN5N11 againstSK-MEL-2 of cancer cell lines.

FIG. 36 represents the anticancer activity of A4W, V8W-GGN5N11 againstA549 of cancer cell lines.

FIG. 37 represents the anticancer activity of A4W, V8W-GGN5N11 againstSK-OV-3 of cancer cell lines.

FIG. 38 represents the anticancer activity of A4W, V8W-GGN5N11 againstHCT116 of cancer cell lines.

FIG. 39 represents the anticancer activity of A4W, V8W-GGN5N11 againstMKN45 of cancer cell lines.

FIG. 40 represents the anticancer activity of A4W, V8W-GGN5N11 againstPC-3 of cancer cell lines.

FIG. 41 represents the anticancer activity of A4W, V8W-GGN5N11 againstA498 of cancer cell lines.

FIG. 42 represents the anticancer activity of A4W, V8W-GGN5N11 againstNCI-H630 of cancer cell lines.

BEST MODE FOR CARRYING OUT THE INVENTION

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

The present invention is more specifically explained by the followingexamples. However, it should be understood that the present invention isnot limited to these examples in any manner.

MODE FOR THE INVENTION

The following Examples and Experimental Examples are intended to furtherillustrate the present invention without limiting its scope.

Example 1 Peptide Synthesis and Purification

To prepare the most optimized antimicrobial and anticancer peptideanalogues showing amphipathic property, novel smaller sizedantimicrobial and anticancer peptides were investigated based on itsparent molecule, i.e., Gaegurin 5 peptide isolated from the skin ofKorean frog (Rana rugosa) according to solid-phase methods usingstandard Fmoc chemistry disclosed in the literature (Wellins D. A. andAtherton, E., Methods EnzymoL, 289, pp 44-67, 1997).

Peptides were synthesized automatically on a peptide synthesizer (Model90, Advanced Chemtech, Inc.). 7 OmM Rink Resins obtained from AdvancedChemtech, Inc. was added to the reaction vessel. The mixture of 2equivalent amount of amino acid, 3 equivalent amount of HOBT(1-Hydroxybenzotrizole) and 2 equivalent amount of DIC(1,3-Diisopropylcarbodiimide) to the amino acid was added to amino acidvessel and dissolved in 10 ml of DMF (Dimethylformamide). The resin inthe reaction vessel was subjected to swelling procedure using by DMFsolvent. The Fmoc (9-fluorenylmethoxycarbonyl) moiety in the resin wasremoved by using 25% piperidine/DMF mixture solvent. The dissolved aminoacid in the amino acid vessel was transferred to a reaction vessel andreacted with resin for 2 or 3 hrs. To link the amino acid with severalamino acids, the above-described step was performed repeatedly to linkwith new amino acid side by side and the Fmoc residue at N-terminalmoiety of the last amino acid was removed by using 25% piperidine/DMFmixture solvent. The protecting groups attached to lysine or serineresidue were eliminated and reacted with 20 ml of 10% TFA (Trifluoroacetic acid)/DCM for 4 hrs to isolate the synthesized peptides fromresin. The solid resin was filtered from the solution and the filteredsolution was added to round flask to distillate. After removing thesolvent, 10 ml of 20% acetonitrile containing 0.1% TFA was added theretoto re-dissolve and remaining solution was filtrated using by centrifugeor filter paper. The supernatant was lyophilized and subjected topurification using by analytical reverse phase HPLC (WE J55B5, HITACHI)on C-18 column (eluting solution: mixture solution of acetonitrile andwater containing 0.1% trifluoroacetic acid, fluid velocity: 1 ml/min)for 45 mins to collect detected fractions comprising only purposedpeptides. The peptides were subjected to lyophilization to obtainpurposed synthesized peptide analogues derived from Gaegurin 5, i.e.,model 25 (F-L-G-W-L-F-K-W-A-K-K), model 26 (F-L-K-W-L-F-K-W-A-K-K),model 27 (F-L-G-W-L-F-K-W-A-W-K) and model 28 (F-L-W-W-L-F-K-W-A-W-K).

Experimental Example 1 Determination of Antimicrobial Activity

To determine the antimicrobial effect of the analogues prepared byExample 1 on the antimicrobial activity, following experiments wereperformed.

1-1. Determination of MIC

Antimicrobial activity was determined by the standard brothmicrodilution method by measuring the MIC values against diversemicroorganisms. In brief, Luria-Bertani medium was used as a brothmedium. 30 μl of sample (2 mg/ml) prepared from Example 1 and 270 μl offluid medium were added to lane 1 well of 96-well microtiter plates andto remaining lanes of the wells, only 150 μl of medium was addedthereto. 150 μl of sample solution in lane 1 was mixed with lane 2 toprepare diluted solution (×2). With similar diluting method to the abovedescribed method, serial dilution was performed to prepare 150 μl ofdiluted drug solution (×2) to the extent that the final drugconcentration reached to the range from 1.6 to 200 μg/ml in respectivewell. 25 μl of the cell culture being grown to 10⁶-10⁸ colony formingunit/ml in 3 ml of broth was added to each well and the micro-titerplate was incubated for overnight at 37° C. The growth of the bacteriawas determined by evaluating UV absorbance of each sample solution at630 nm and the MIC value was determined as the lowest peptideconcentration which completely inhibited the cell growth.

At the result, all the model peptide analogues showed potentantimicrobial activity for various strains compared to that of Gaegurin5 as can be shown in Table 1.

1-2. Determination of Hemolysis

Hemolytic activities of peptide analogues were measured as follows;

3 ml of blood sample collected from healthy male was mixed with PBS(phosphate buffered saline, isotonic solution) with a ratio of 1:1(v/v), centrifuged to remove buffy coat and blood plasma and washed withphysiological saline solution three times to isolate pure erythrocyte.The erythrocyte was suspended in 20 ml of PBS and pre-incubated in waterbath at 37° C. for 15 mins. 10 ml of peptide solution was mixed with 190ml of erythrocyte solution in order to prepare various mixed solutioncontaining various final concentrations of the peptides, i.e., 100, 50,25 mg/ml and the solution was incubated in water bath at 37° C. for 15mins. The supernatant was centrifuged with centrifuge and 100 ml of thesupernatant was diluted with ImI of PBS to determine its absorbance at550 nm. The percentage of hemolysis (%) was determined from the relativeattenuation of the absorbance of sample compared with that of thesuspension treated with 0.2% Triton X-100.

At the result, model peptide analogues showed mere hemolytic activitysimilar to their parent molecule, i.e., Gaegurin 5 as can be shown inTable 1. Accordingly, it is confirmed that the peptide analogues of thepresent invention are safe and suitable to drug development,particularly model 26; which showed much less hemolysis than the others.

TABLE 1 model model model model W4,8- 25 26 27 28 GGN5N11 Minimalinhibitory concentration (μl/ml) Microorganism Gram-positive bacteriaBacillus subtilis 6.3 3.2 6.3 3.2 3.2 Staphylococcus aureus 3.2 3.2 3.23.2 1.6 Staphylococcus 6.3 6.3 6.3 3.2 3.2 epidermis Micrococcus luteus6.3 6.3 6.3 3.2 3.2 Propionibacterium — 3.2 — — — acnes ATCC 3314Propionibacterium — 6.3 — — — acnes ATCC 3320 Propionibacterium — 3.2 —— — acnes ATCC 5012 Gram negative bacteria Escherichia coli 12.5 12.525 >200 50 Shigella dysentariae 25 25 12.5 >200 12.5 Salmonella 50 50100 >200 >200 typhimurium Klebsiella pneumoniae 12.5 12.5 12.5 >200 50Proteus mirabilis >200 >200 >200 >200 >200 Pseudomonas 2512.5 >200 >200 >200 aeruginosa Hemolysis values (%) Peptideconcentration 100 μg/ml 19.41 16.26 20.28 19.37 26.20

Experimental Example 2 Determination of Anticancer Activity

To determine the inhibiting activity of the analogues prepared inExample 1 on the cancer cell growth, following experiment was performed.

In order to confirm the anticancer activity of the analogues prepared byExample 1 on A549 (lung cancer cell line, ATCC, U.S.A.), A498 (kidneycancer cell line, Korean Cell Line Bank, Korea), HCT116 (colon carcinomacell line, Korean Cell Line Bank, Korea), MKN45 (stomach cell line,Korean Cell Line Bank, Korea), NCI-H630 (liver cancer cell line, KoreanCell Line Bank, Korea), PC-3 (prostatic carcinoma cell line, Korean CellLine Bank, Korea), SK-MEL-2 (skin cancer cell line, Korean Cell LineBank, Korea) and SK-OV-3 (human solid cancer cell line, Korean Cell LineBank, Korea), the standard microculture MTT method was performed withthe procedure described in the literature (Angelina Quintero et al., JPharm Pharmaceut Sci, Vol. 2, No. 3, pp 108-112, 1999; Ashtosh K. Pathaket al., J. Am Coll Nutr., Vol. 21, No. 5, pp 416-421, 2002).

Each cell line was poured into 96 well microtiter plates and cultivatedat 37° C. for 24 hrs. When the cell reached to exponential growth phase,the test sample was added to each plates and each cell was inoculatedinto the plates added with both of dissolved and non-dissolved DMSO toculture for 3 days. 50 μl of MTT (2 mg/ml) (M5655, SIGMA, U.S.A.) wasadded thereto and cultured for 3 hrs. The supernatant was removed andthe wells were shaken weakly to dissolve formazan crystals. 150 μl ofDMSO was added thereto. The non-treated well with cell was set to blankand the wells non-treated with drug were set to standard based on thecell viability. The UV absorbance was measured by microplate reader(SAFIRE, Tecan, Austria) to calculate the cell viability at 570 nm. Theinhibition rate of cell growth was calculated by following Math FIG. 1.

inhibition rate of cell growth (%)={1−(absorbance of treatedcells/absorbance of non-treated cells)}×100  Math Figure 1

The IC value of each cell was calculated by the amount of drug causingto 50% reduction of the absorbance comparing with that of non-treatedcells.

At the result, all the model peptide analogues showed anticanceractivity similar to their parent molecule, i.e., Gaegurin 5 as can beshown in Table 2 and FIGS. 7 to 42.

TABLE 2 IC₅₀: mM Tumor cell lines model 25 model 26 model 27 model 28A498 41.21 21.48 n.a. n.a. A549 25.2 14.53 20.36 15.88 HCT116 27 14.824.63 14.8 MKN45 26.7 22.51 24.31 13.77 NCI-H630 n.a. n.a. 48 57 PC-347.69 29.1 46.51 24.32 SK-MEL-2 27.39 22.25 13.97 13.16 SK-OV-3 24.7612.55 22.06 14.58 n.a.: not available

Hereinafter, the formulating methods and kinds of excipients will bedescribed, but the present invention is not limited to them. Therepresentative preparation examples were described as follows.

Preparation of Powder

Peptide analogue of Example 1 50 mg Lactose 100 mg  Talc 10 mg

Powder preparation was prepared by mixing above components and fillingsealed package.

Preparation of Tablet

Peptide analogue of Example 1  50 mg Corn Starch 100 mg Lactose 100 mgMagnesium Stearate  2 mg

Tablet preparation was prepared by mixing above components andentabletting.

Preparation of Capsule

Peptide analogue of Example 1  50 mg Corn Starch 100 mg Lactose 100 mgMagnesium Stearate  2 mg

Capsule preparation was prepared by mixing above components and fillinggelatin capsule by conventional gelatin preparation method.

Preparation of Injection

Peptide analogue of Example 1 50 mg Distilled water for injectionoptimum amount PH controller optimum amount

Injection preparation was prepared by dissolving active component,controlling pH to about 7.5 and then filling all the components in 2 mlampule and sterilizing by conventional injection preparation method.

Preparation of Liquid

Peptide analogue of Example 1 0.1-80 g Sugar   5-10 g Citric acid0.05%-0.3%    Caramel 0.005-0.02%  Vitamin C 0.1-1%   Distilled water79-94% CO₂ gas  0.5-0.82%

Liquid preparation was prepared by dissolving active component, fillingall the components and sterilizing by conventional liquid preparationmethod.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

The analogues of antimicrobial and anticancer peptide synthesized andproduced from Gaegurin 5 of the present invention show potentantimicrobial activity against gram positive and negative strains,potent anticancer activity against eight kinds of cancer cell lines,good safety with very low hemolytic activity and favorable advantagessuch as drug absorption and drug transportation due to its advantageousstructural property, i.e., the shortest structure among previously knownantimicrobial and anticancer peptides.

SEQUENCE LISTING

SEQ ID NO. 1: F-L-G-W-L-F-K-W-A-K-K is “model 25” synthesized fromGaegurin 5, SEQ ID NO. 2: F-L-K-W-L-F-K-W-A-K-K is “model 26”synthesized from Gaegurin 5, SEQ ID NO. 3: F-L-G-W-L-F-K-W-A-W-K is“model 27” synthesized from Gaegurin 5, SEQ ID NO. 4:F-L-W-W-L-F-K-W-A-W-K is “model 28” synthesized from Gaegurin 5.

1. Analogues of antimicrobial and anticancer peptide represented by Seq.I.D. 1 (F-L-G-W-L-F-K-W-A-K-K) synthesized and produced from Gaegurin 5.2. Analogues of antimicrobial and anticancer peptide represented by Seq.I.D. 2 (F-L-K-W-L-F-K-W-A-K-K) synthesized and produced from Gaegurin 5.3. Analogues of antimicrobial and anticancer peptide represented by Seq.I.D. 3 (F-L-G-W-L-F-K-W-A-W-K) synthesized and produced from Gaegurin 5.4. Analogues of antimicrobial and anticancer peptide represented by Seq.I.D. 4 (F-L-W-W-L-F-K-W-A-W-K) synthesized and produced from Gaegurin 5.5. The analogues of claim 1 wherein said peptide is synthesized by amethod selected from the group consisting of substituting amphipathicboundary surface of the peptide with tryptophan and substitutingamphipathic boundary surface of the peptide with lysine.
 6. (canceled)7. A pharmaceutical composition comprising said antimicrobial andanticancer peptide as set forth in claim 1 to as an effective ingredientand pharmaceutically acceptable carrier or adjuvant for the treatmentand prevention of infective diseases caused by a microorganism.
 8. Thepharmaceutical composition of claim 7 wherein said infective diseasescaused by microorganism is selected from the group consisting ofstaphylococcus food poisoning, cellulitis, urinary tract infection,meningitis, peritonitis, cystitis, lymphangitis, felon, tympanitis,respiratory disease, pneumonia, purulent inflammation and sepsis.
 9. Apharmaceutical composition comprising said antimicrobial and anticancerpeptide as set forth in claim 1 as an effective ingredient andpharmaceutically acceptable carrier or adjuvant for the treatment andprevention of cancer diseases.
 10. The pharmaceutical composition ofclaim 9 wherein said cancer diseases is selected from the groupconsisting of cervical cancer, lung cancer, pancreas cancer, non-smallcell lung cancer, liver cancer, colon carcinoma, cancer of a bone, skincancer, cancer of the head and neck, cutaneous or intraocular melanoma,uterine carcinoma, ovarian cancer, rectal cancer, stomach cancer, cancerof the anal region, breast cancer, oviduct cancer, endometrialcarcinoma, vaginal cancer, vulva cancer, Hodgkin's disease, esophagealcancer, small intestine tumor, endocrine gland's cancer, thyroid cancer,parathyroid cancer, epinephros cancer, soft tissue sarcomas,urethrophyma, penis cancer, prostatic carcinoma, bladder cancer, kidneyand ureter cancer.
 11. A method of treating or preventing the infectivediseases caused by microorganism and cancer diseases of human andmammals comprising administering an effective amount of saidantimicrobial and anticancer peptide as set forth in claim 1 synthesizedand produced from Gaegurin 5 together with a pharmaceutically acceptablecarrier thereof.
 12. A use of the composition comprising saidantimicrobial and anticancer peptide as set forth in claim 1 synthesizedand produced from Gaegurin 5, for the manufacture of a medicament forinfective diseases caused by microorganism and cancer diseases in amammal.
 13. The analogues of claim 2 wherein said peptide is synthesizedby a method selected from the group consisting of substitutingamphipathic boundary surface of the peptide with tryptophan andsubstituting amphipathic boundary surface of the peptide with lysine.14. A pharmaceutical composition comprising said antimicrobial andanticancer peptide as set forth in claim 2 as an effective ingredientand pharmaceutically acceptable carrier or adjuvant for the treatmentand prevention of infective diseases caused by a microorganism.
 15. Thepharmaceutical composition of claim 14 wherein said infective diseasescaused by microorganism is selected from the group consisting ofstaphylococcus food poisoning, cellulitis, urinary tract infection,meningitis, peritonitis, cystitis, lymphangitis, felon, tympanitis,respiratory disease, pneumonia, purulent inflammation and sepsis.
 16. Apharmaceutical composition comprising said antimicrobial and anticancerpeptide as set forth in claim 2 as an effective ingredient andpharmaceutically acceptable carrier or adjuvant for the treatment andprevention of cancer diseases.
 17. The pharmaceutical composition ofclaim 16 wherein said cancer diseases is selected from the groupconsisting of cervical cancer, lung cancer, pancreas cancer, non-smallcell lung cancer, liver cancer, colon carcinoma, cancer of a bone, skincancer, cancer of the head and neck, cutaneous or intraocular melanoma,uterine carcinoma, ovarian cancer, rectal cancer, stomach cancer, cancerof the anal region, breast cancer, oviduct cancer, endometrialcarcinoma, vaginal cancer, vulva cancer, Hodgkin's disease, esophagealcancer, small intestine tumor, endocrine gland's cancer, thyroid cancer,parathyroid cancer, epinephros cancer, soft tissue sarcomas,urethrophyma, penis cancer, prostatic carcinoma, bladder cancer, kidneyand ureter cancer.
 18. A method of treating or preventing the infectivediseases caused by microorganism and cancer diseases of human andmammals comprising administering an effective amount of saidantimicrobial and anticancer peptide as set forth in claim 2 synthesizedand produced from Gaegurin 5 together with a pharmaceutically acceptablecarrier thereof.
 19. A use of the composition comprising saidantimicrobial and anticancer peptide as set forth in claim 2 synthesizedand produced from Gaegurin 5, for the manufacture of a medicament forinfective diseases caused by microorganism and cancer diseases in amammal.
 20. The analogues of claim 3 wherein said peptide is synthesizedby a method selected from the group consisting of substitutingamphipathic boundary surface of the peptide with tryptophan andsubstituting amphipathic boundary surface of the peptide with lysine.21. A pharmaceutical composition comprising said antimicrobial andanticancer peptide as set forth in claim 3 as an effective ingredientand pharmaceutically acceptable carrier or adjuvant for the treatmentand prevention of infective diseases caused by a microorganism.
 22. Thepharmaceutical composition of claim 21 wherein said infective diseasescaused by microorganism is selected from the group consisting ofstaphylococcus food poisoning, cellulitis, urinary tract infection,meningitis, peritonitis, cystitis, lymphangitis, felon, tympanitis,respiratory disease, pneumonia, purulent inflammation and sepsis.
 23. Apharmaceutical composition comprising said antimicrobial and anticancerpeptide as set forth in claim 3 as an effective ingredient andpharmaceutically acceptable carrier or adjuvant for the treatment andprevention of cancer diseases.
 24. The pharmaceutical composition ofclaim 23 wherein said cancer diseases is selected from the groupconsisting of cervical cancer, lung cancer, pancreas cancer, non-smallcell lung cancer, liver cancer, colon carcinoma, cancer of a bone, skincancer, cancer of the head and neck, cutaneous or intraocular melanoma,uterine carcinoma, ovarian cancer, rectal cancer, stomach cancer, cancerof the anal region, breast cancer, oviduct cancer, endometrialcarcinoma, vaginal cancer, vulva cancer, Hodgkin's disease, esophagealcancer, small intestine tumor, endocrine gland's cancer, thyroid cancer,parathyroid cancer, epinephros cancer, soft tissue sarcomas,urethrophyma, penis cancer, prostatic carcinoma, bladder cancer, kidneyand ureter cancer.
 25. A method of treating or preventing the infectivediseases caused by microorganism and cancer diseases of human andmammals comprising administering an effective amount of saidantimicrobial and anticancer peptide as set forth in claim 3 synthesizedand produced from Gaegurin 5 together with a pharmaceutically acceptablecarrier thereof.
 26. A use of the composition comprising saidantimicrobial and anticancer peptide as set forth in claim 3 synthesizedand produced from Gaegurin 5, for the manufacture of a medicament forinfective diseases caused by microorganism and cancer diseases in amammal.
 27. The analogues of claim 4 wherein said peptide is synthesizedby a method selected from the group consisting of substitutingamphipathic boundary surface of the peptide with tryptophan andsubstituting amphipathic boundary surface of the peptide with lysine.28. A pharmaceutical composition comprising said antimicrobial andanticancer peptide as set forth in claim 4 as an effective ingredientand pharmaceutically acceptable carrier or adjuvant for the treatmentand prevention of infective diseases caused by a microorganism.
 29. Thepharmaceutical composition of claim 28 wherein said infective diseasescaused by microorganism is selected from the group consisting ofstaphylococcus food poisoning, cellulitis, urinary tract infection,meningitis, peritonitis, cystitis, lymphangitis, felon, tympanitis,respiratory disease, pneumonia, purulent inflammation and sepsis.
 30. Apharmaceutical composition comprising said antimicrobial and anticancerpeptide as set forth in claim 4 as an effective ingredient andpharmaceutically acceptable carrier or adjuvant for the treatment andprevention of cancer diseases.
 31. The pharmaceutical composition ofclaim 30 wherein said cancer diseases is selected from the groupconsisting of cervical cancer, lung cancer, pancreas cancer, non-smallcell lung cancer, liver cancer, colon carcinoma, cancer of a bone, skincancer, cancer of the head and neck, cutaneous or intraocular melanoma,uterine carcinoma, ovarian cancer, rectal cancer, stomach cancer, cancerof the anal region, breast cancer, oviduct cancer, endometrialcarcinoma, vaginal cancer, vulva cancer, Hodgkin's disease, esophagealcancer, small intestine tumor, endocrine gland's cancer, thyroid cancer,parathyroid cancer, epinephros cancer, soft tissue sarcomas,urethrophyma, penis cancer, prostatic carcinoma, bladder cancer, kidneyand ureter cancer.
 32. A method of treating or preventing the infectivediseases caused by microorganism and cancer diseases of human andmammals comprising administering an effective amount of saidantimicrobial and anticancer peptide as set forth in claim 4 synthesizedand produced from Gaegurin 5 together with a pharmaceutically acceptablecarrier thereof.
 33. A use of the composition comprising saidantimicrobial and anticancer peptide as set forth in claim 4 synthesizedand produced from Gaegurin 5, for the manufacture of a medicament forinfective diseases caused by microorganism and cancer diseases in amammal.